Biocompatibility of Intracanal Medications Based on Calcium Hydroxide

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Volume 2012, Article ID 904963,6pages doi:10.5402/2012/904963

Research Article

Biocompatibility of Intracanal Medications Based on

Calcium Hydroxide

Carolina Andolfatto,

1

_{Guilherme Ferreira da Silva,}

1

_{Ana Livia Gomes Corn´elio,}

1

Juliane Maria Guerreiro-Tanomaru,

1

_{Mario Tanomaru-Filho,}

1

_{Gisele Faria,}

1

Idomeo Bonetti-Filho,

1

_{and Paulo S´ergio Cerri}

2

1_{Department of Restorative Dentistry, Araraquara Dental School, Universidade Estadual Paulista (UNESP),}

14801-903 Araraquara, SP, Brazil

2_{Laboratory of Histology and Embryology, Department of Morphology, Araraquara Dental School,}

Universidade Estadual Paulista (UNESP), 14801-903 Araraquara, SP, Brazil

Correspondence should be addressed to Paulo S´ergio Cerri,[email protected]

Received 6 November 2012; Accepted 27 November 2012

Academic Editors: H. S. Cardash, J. H. Jeng, and G. Mount

Copyright © 2012 Carolina Andolfatto et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Objective. The aim of this study was to evaluate the rat subcutaneous tissue reaction to calcium hydroxide-based intracanal

medicaments, UltraCal XS (calcium hydroxide, barium sulphate, aqueous matrix), Hydropast (calcium hydroxide, barium sulphate, and propyleneglycol), and Calen (Calcium hydroxide, zinc oxide, colophony, and polyethyleneglycol), used as a control.

Methods. Forty-eight rats (Rattus Norvegicus Holtzman) were distributed in three groups: Calen, UltraCal XS, and Hydropast.

Polyethylene tubes filled with one of the medicaments were implanted in the dorsal subcutaneous. After 7 and 30 days, the implants were removed and the specimens were fixed and embedded in paraffin. Morphological and quantitative analyses were carried out in the HE-stained sections. The numerical density of inflammatory cells in the capsule was evaluated and statistical analyses were performed (P≤0.05).Results. At 7 days, all materials induced an inflammatory reaction in the subcutaneous tissue adjacent to the implants. In all groups, a significant reduction in the number of inflammatory cells and giant cells was verified in the period of 30 days.Conclusion. These results indicate that the calcium hydroxide-based medicaments evaluated present biocompatibility similar to Calen.

1. Introduction

The success of endodontic treatment of teeth with periapical lesion depends on the reduction or elimination of the

intraradicular infection [1, 2]. The root canal mechanical

preparation is not enough to eliminate this infection because many microorganisms are not only in the main root canal, but also disseminated throughout the root canal system. Therefore, the use of an intracanal dressing to eliminate the microorganisms is indicated [3–6].

Antimicrobial activity and biocompatibility are charac-teristics that an ideal intracanal dressing has to show [7]. Calcium hydroxide [Ca(OH)2] has been widely used for its

biological and antimicrobial activity [4,8], ability to dissolve

organic tissue [9], and capacity to inactivate bacterial

endotoxin [10,11]. Despite these properties, the Ca(OH)2

has no satisfactory physical properties such as radiopacity to visualize on dental radiographs and flow capacity to facilitate

its insertion in the root canal [12, 13]. For this reason, it

needs the incorporation of a radiopacifying agent and a

vehicle to improve these characteristics [8,14].

Although the Ca(OH)2 shows an excellent

biocom-patibility, the addition of other substances can affect its

biological properties [8,12]. In the last years, it has been

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demonstrated that UltraCal XS has a high pH value [15]

and an effective antimicrobial activity against common

endodontic bacteria of teeth with pulp necrosis [16]. On the other hand, Brazilian paste Hydropast, composed by 38% of calcium hydroxide, barium oxide, as a radiopacifying agent, and propilenoglycol as vehicle, is a recent material, and, therefore, until now, there are no studies of its biological properties.

Considering the recommendation of International

Orga-nization for Standardization [17], it is necessary in vitro

and/orin vivostudies for evaluation of the biocompatibility

of these new materials. Thus, the purpose of this study was to evaluate the tissue reaction of these calcium hydroxide-based medicaments in rat subcutaneous.

2. Materials and Methods

2.1. Animals and Experimental Proceedings. This study was

performed in accordance with the principles of animal care on animal experiments. The research protocol was autho-rized by the Ethical Committee for Animal Research of the S˜ao Paulo State University, Brazil (Dental School, UNESP, Araraquara).

Forty-eight male Holtzman rats (Rattus norvegicus

albi-nus), weighing 250±10 g, were kept in individual stainless

steel cages under 12 : 12 light-dark cycle at controlled

temperature (23±2◦_{C) and humidity (55 + 10%), with food}

and water providedad libitum. The animals were randomly

distributed into three groups (n = 16) on the basis of the

intracanal medicaments analysed, Calen group, Calen paste (S. S. White Artigos Dentários Ltda., Rio de Janeiro, RJ, Brazil), used as control group; UltraCal group, UltraCal XS paste (Ultradent Products, Inc., South Jordan, UT, USA), and Hydropast group, Hydropast (Biodinâmica Qu´ımica e Farmacêutica Ltda., Ibipora, PR, Brazil). The composition of

these materials is described at Table1.

The polyethylene tubes (Embramed Ind. Com. Ltda., S˜ao Paulo, SP, Brazil) with 10.0 mm length and 1.5 mm diameter previously sterilized with ethylene oxide were filled with Calen, UltraCal XS paste, or Hydropast intracanal medicaments.

The animals were anaesthetized with an intraperitoneal injection containing 80 mg/Kg of body weight of ketamine (União Qu´ımica Farmacêutica Nacional S/A-Brazil) and 4 mg/Kg of body weight of xylazine (Virbac do Brasil Ind ústria e Comércio Ltda., Brazil). After shaved and disinfection with 5% iodine solution, a 20 mm-long incision in a head-to-tail orientation was made using a scalpel (no. 15, Fibra Cir úrgica, Joinvile, SC, Brazil) in the dorsal skin. Subsequently, the polyethylene tube containing Calen paste, UltraCal XS, or Hydropast was immediately implanted into the dorsal subcutaneous connective tissue. After implan-tation, the skin was closed with 4.0 silk suture (Vicryl; Johnson & Johnson: Ethicon Inc., New Brunswick, NJ, USA). One polyethylene tube filled with an intracanal paste was implanted in each animal and left for periods of 7 and 30 days.

Table_{1: Tested materials and composition.}

Composition Manufacturer

Calen

49.77% calcium hydroxide, 50.23% of zinc oxide, colophony, and polyethylene glycol 400

S. S. White Artigos Dent´arios Ltda., Rio de Janeiro, RJ, Brazil

UltraCal XS 35% calcium hydroxide_{and a radiopacifier}

Ultradent Products, Inc., South Jordan, UT, USA

Hydropast

38% of calcium hydroxide, 62% of barium oxide, and propilenoglicol

Biodinamica Quimica e Farmaceutica Ltda., Ibipora, PR, Brazil

After experimental periods, the animals were killed by overdose of anesthetic solution, and the tubes were removed

with surrounding connective tissue and prepared for paraffin

embedding.

2.2. Histological Procedures and Analysis. The specimens

containing the implanted polyethylene tubes were fixed in 4% formaldehyde (prepared from paraformaldehyde)

buffered at pH 7.2 with 0.1 M sodium phosphate at room

temperature for 48 hours. Subsequently, the specimens were

dehydrated and embedded in paraffin. Serial 6µm-thick

sections were made parallel to the tube long axis and stained with hematoxylin and eosin (H&E) for morphological and morphometric analyses. The morphological analysis of the capsule in contact with the material on the opening of the tube was performed considering the following parameters: presence of inflammatory process, main cells (inflammatory cells or fibroblasts) present in the capsule, presence of multinucleated giant cells, and presence of collagen fibers.

The numerical density of inflammatory mononucleated cells and multinucleated giant cells was undertaken using a light microscope (BX51, Olympus, Tokyo, Japan) and an image analysis system (Image Pro-Express 6.0, Olympus). Three H&E-stained sections per animal were selected at

intervals of at least 100µm; in each section, a standardized

field of 0.09 mm2 _{of the connective tissue adjacent to the}

opening of the tube implanted was analyzed, totaling

0.27 mm2 _{per animal. In each area, the total number of}

inflammatory cells was counted using the image analysis

sys-tem at×40 magnification; in each animal, the total number

of inflammatory cells was divided by total area, and, then,

the number of inflammatory cells/mm2 _{was obtained. The}

differences between the groups were statistically analyzed

using SigmaStat 2.0 software (Jandel Scientific, Sausalito, CA, USA); the data were submitted to ANOVA and Tukey test.

The significance level accepted wasP≤0.05.

3. Results

3.1. Morphological and Quantitative Analyses. After 7 days

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I BV BV CG

CG

IC

I

Fb _CF

Fb

CF

BV BV

(a)

(b)

(c)

Figure_{1: Light micrographs of sections showing portions of capsules adjacent to the opening of the tubes (I) filled with Calen paste after 7}

(a) and 30 days (b and c) of implantation in the subcutaneous. In (a), numerous inflammatory cells (ICs) are observed in the inner portion of the capsule adjacent to the tube opening (I). The inset of the outlined area shows multinucleated giant cells (GCs) in close juxtaposition to the material implanted. BV, blood vessels.×110; inset:×250 (b) shows the capsule exhibits several fibroblasts (Fb) dispersed among

the collagen fiber bundles (CF)×130. In (c), outlined area in (b), shows some inflammatory cells (arrows), mainly lymphocytes, situated adjacent to the blood vessels (BV)×250.

I BV

BV

I IC

IC CG

BV

IC

(a)

I BV

BV

FC Fb

Fb

(b)

Figure_{2: Light micrographs of sections showing portions of capsules adjacent to the opening of the tubes (I) filled with UltraCal XS paste}

after 7 (a) and 30 days (b) of implantation in the subcutaneous. In (a), The capsule exhibits several inflammatory cells (IC) and blood vessels (BV). Note that dense masses of inflammatory cells (ICs) are observed in the inner portion of the capsule, adjacent to the tube opening (I). The inset, outlined area, shows multinucleated giant cells (GCs).×120; inset:×230. (b) The capsule contains several fibroblasts (Fbs) and numerous blood vessels (BV). Inflammatory cells (arrows), lymphocytes and macrophages are situated mainly in the inner portion of the capsule and next to the blood vessels×150.

evident in the innermost portion of the capsule, that is, in

close juxtaposition to the materials (Figures1(a),2(a)and

3(a)). According to Table 2, no significant difference was

verified in the numerical density of inflammatory cells among the groups. Multinucleated giant cells were also observed in the capsules formed in all groups (Figures

1(a),2(a), and3(a)). However, in the Hydropast group the

number of multinucleated giant cells was significantly higher

in comparison to other groups; on the other hand, the capsule of the Calen group exhibited the lower number of

multinucleated giant cells (Table2).

A significant decrease in the number of inflammatory cells and multinucleated giant cells was verified from 7 to

30 days, in all groups (Figures1(b),2(b), and3(b); Table2).

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I N

IC

IC BV

CG CG

(a)

I CF

BV

CF

BV

FC BV

(b)

Figure_{3: Light micrographs of sections showing portions of capsules adjacent to the opening of the tubes (I) filled with Hydropast after 7}

(a) and 30 days (b) of implantation in the subcutaneous. In (a), numerous inflammatory cells (ICs) and multinucleated giant cells (GCs) are present by thorough capsule. BVs, blood vessels×120. (b) The capsule contains predominantly inflammatory cells (arrows); scarce collagen

fibers (FC) distributed irregularly are observed in the capsule. BVs, blood vessels×120.

Table_{2: Number of inflammatory cells and giant cells/mm}2_{of the capsule adjacent to the implants in the subcutaneous.}

Inflammatory cells Giant cells

7 days 30 days 7 days 30 days

Calen 2,795.10±939.90a,1 _960.64_±_433.22a,2 _4.14_±_0.90a,1 _0.92_±_0.18a,2

UltraCal XS 2,397.66±613.14a,1 975.99±227.71a,2 6.00±1.03b,1 2.77±1.05b,2

Hydropast 2,248.10±315.41a,1 1,407.82±870.89a,2 8.77±1.46c,1 1.37±0.87c,2

Values are expressed as mean±standard deviation.

Equal letters indicate no statistically significant difference (P >0.05) between the materials in the same experimental period. Different numbers indicate difference statistically significant (P≤0.05) of each material in the different experimental periods.

bundles of collagen fibers between fibroblasts; lymphocytes and plasma cells were mainly present next to blood vessels

(Figure1(b)). In the UltraCal group, the capsule contained

several fibroblasts among the inflammatory cells; usually, bundles of collagen fibers were only present in the outermost

portion of the capsule (Figure2(b)). The connective tissue

of the capsule of the Hydropast exhibited several cells and

scarce collagen fibers (Figure3(b)). Although no significant

difference was found in the number of inflammatory cells

between the groups, the mean of the numerical density of inflammatory cells in the Hydropast group was around

1,400 cells/mm2_{, whereas in the other groups was around}

965 cells/mm2_{. Moreover, in the Calen group the number}

of multinucleated giant cells was significantly lower in

comparison to UltraCal and Hydropast groups (Table2).

4. Discussion

Implantation in the subcutaneous connective tissues of experimental animals has been extensively used to evaluate

the biocompatibility of endodontic materials [18,19]. Our

findings indicate that UltraCal XS and Hydropast exhibit biological behavior similar to Calen (control group). At 7 days, an intense inflammatory reaction and foci of coag-ulative necrosis were seen in the adjacent capsule to the implanted materials. After 30 days, significant reduction

in the inflammatory process was verified in all analyzed groups; usually, the capsule formed juxtaposed to the Calen paste exhibited inflammatory cells among typical bundles of collagen fibers and fibroblasts. The inflammatory reaction observed in the period of the 7 days may be attributed to the superficial necrosis promoted by calcium hydroxide-based materials [20]. Calcium hydroxide has an alkaline pH [21] and, when in contact with the connective tissue, induces the formation of a coagulative necrosis zone [22]. Coagulative necrosis refers to a spectrum of morphological changes in living tissue resulting from the action of enzymes on lethally injured cells. The mass of necrotic cells is characterized by preservation of the basic outline of the coagulated cells for a span of at least some days. As necrotic cells are unable to maintain membrane integrity, their contents leak out and elicit an inflammatory response that removes the cellular debris by phagocytosis, followed by healing [20].

The vehicles mixed with calcium hydroxide powder play an important role in the ionic dissociation process and so in the disinfection of the root canal and biocompatibility

[8,23]. There are three main types of vehicle: water-soluble

substances, viscous, and oil-based [8]. In the present study,

the vehicles of the different medicaments did not interfere

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to Hydropast showed a significantly lower number of giant cells than the UltraCal XS. The giant cells are derived from the fusion of 20 or more monocytes/macrophages and are formed for removing exogenous agents [24]. In the period of 30 days, the high number of giant cells verified in the UltraCal XS group suggests that this material may release more irritant substances than the Hydropast and Calen.

In Calen paste, the calcium hydroxide is mixed to a viscous vehicle, polyethylene glycol 400, one of the most commonly used vehicles in root canal medicaments with low toxicity, high solubility in aqueous solutions, low immuno-genicity and antiimmuno-genicity [25], and antibacterial activity [5]. This vehicle releases calcium and hydroxyl ions more slowly and for longer periods than water-soluble and oil-based

materials [26]. Because the releasing of H+_{, the polyethylene}

glycol 400 neutralizes the OH−_{released by calcium hydroxide}

and, thereby, reduces the superficial necrosis area [27]. The propylene glycol is used as vehicle in the Hydropast; the vehicle used in the Hydropast is classified as a viscous vehicle with high molecular weight and, as well as polyethy-lene glycol 400, prolong the action of calcium hydroxide in the root canal system [26]. Moreover, propylene glycol shows

low toxicity and antimicrobial properties [28, 29]. It was

demonstrated that the addition of propylene glycol may not interfere in the biocompatibility of MTA in rat subcutaneous tissue [30].

Our results also suggest that the different radiopacifying

agents of the pastes did not interfere in the tissue reaction.

The endodontic materials should present sufficient

radiopac-ity to be distinguished from adjacent anatomical structures, such as bone and teeth [31]. Zinc oxide, barium sulfate, bismuth oxide, and other components with iodine and

bromine are some examples of radiopacifiers [12,14,27,32].

The zinc oxide of Calen paste does not a_ffect the

biological properties of calcium hydroxide [27,33]. Barium

sulphate, radiopacifying agent of Hydropast, is also

bio-compatible because its cause no detrimental effect in rat

subcutaneous tissue [34] or in periapical tissue in association with calcium hydroxide [14]; barium oxide in association with the Norian, an skeletal repair system (SRS), in tibiae defects of rats, maintains the properties of biocompatibility and osteoconductive materials of the SRS [35]. Although the manufacturer does not inform the radiopacifying agent of the UltraCal XS, our results demonstrated that this material has a good biological behavior.

Considering the methodology used in the present study, our findings indicate that UltraCal XS and Hydropast are biocompatible in subcutaneous tissue of rats.

Conflict of Interests

The authors declared that they have no any conflict of inte-rests related to this study.

Acknowledgments

The authors thank Mr. Pedro Sérgio Simões for technical support. This paper was supported by Coordenação de

Aperfeic¸oamento de Pessoal de N´ıvel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol ´ogico (CNPq), Brazil.

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